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Isolation of Bioluminescent Bacteria and Their Application in Toxicity Testing of Chromium in Water

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Isolation of Bioluminescent Bacteria and Their Application in Toxicity Testing of Chromium in Water Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 23-32 ISSN: 2319-7706 Volume 4 Number 10 (2015) pp. 23-32 http://www.ijcmas.com Original Research Article Isolation of Bioluminescent Bacteria and Their Application in Toxicity Testing of Chromium in Water Varsha Shivaji Kulkarni* and Bhagyashree Shashikant Kulkarni Department of Microbiology, Sinhgad College of Science, Ambegaon (Bk), Pune-411046, India *Corresponding author A B S T R A C T Nature has its own beauty and phenomenon called bioluminescence proves it. Marine fishes like Japanese Threadfin brin (Nemipterus japonicas), Indian Mackerel (Restrelliger kanagurta) and Anchovy (Engraulis japonicas) were chosen as source of bioluminescent bacteria. Scales, gills and eye portion along with gut region of Indian Mackerel were suspended overnight in sterile sea salt saline and from this loopful suspension were streak inoculated on sterile Photobacterium agar, LA, Sea water agar and Boss Agar. By subsequent subculturing two luminescent isolates K e y w o r d s named as C1, C2 were obtaining on LA plate from the gut portion of the Indian Mackerel. They were used for further study. Morphological and biochemical Biolumine- characterization along with observation of PHB granules under light microscope scence, suggests that the isolates may belong to Genus Vibrio. To reconfirm these results the Luciferase, isolates were inoculated on TCBS agar which is the highly specific media for the Auto- Vibrio identification and confirmation; which confirmed that the isolate C1 may be induction, late sucrose fermenter Vibrio species. This isolate showed green coloured colonies Pollutant after 36hrs of incubation. The TSI slant examination stated that the organisms were chromium, Gram negative enteric pathogen that ferments glucose, lactose and sucrose by acid Toxicity production without accumulation of gas. After examination of growth pattern of both testing isolates; C1 and C2 were used for the checking toxicity of the chemical pollutant cultivation chromium in water. According to the United States Environmental Protection Agency for primary drinking water the standard Maximum Contaminant Level is 0.1 mg/L for total chromium. Standard protocols were used to determine total chromium level in water. Performance of the TVC of the both isolates lead to the results that the pollutant chromium inhibits the growth of bioluminescent bacteria and also affects the luminescence intensity. This whole study provides eco friendly and cost effective examination of pollutant chromium. The method can also be used to measure other chemicals, metal salts like mercuric chloride, zinc chloride and lead acetate etc. Introduction Bioluminescence a Greek word ( Bios for This type of chemiluminescent phenomenon living and Latin word lumen for light ) have been seen in many types of organisms which literally means a living light. (bacteria, fungi, fishes, squid, 23 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 23-32 dinoflagellates) and have always gained an bacteria and algae gaining the attention for a attention of scientists because of their toxicity evaluation as a model organisms beauty, physiology, biochemistry, genetics (Roda et al., 2004; Boynton, 2009). As well and ease of detection (Lee 2008; known; general metabolism, operon Vedprakash et al., 2014). In late 19th century expression, bacterial growth, cultural Raphael Dubois experimentally extracted condition affects the luminescence activity; the two key components, enzyme from which one can articulate that the luciferase and luciferine of bioluminescent organisms are very much bioluminescence reaction which are able to sensitive to any change (e.g. physical, generate light (Makemson, 1986; Lee, chemical, radiological, etc.) in environment. 2008). Many bacteria regulate their set of Bioluminescent bacterial tests have been lux genes by the mechanism of quorum successfully used previously in determining sensing or autoinduction in which the toxicity of aquatic samples, sediments, autoinducers (AI) evoke the characteristic and soils (Lapota et al., 1998; Kurvet et al., response from the cell (Rees et al., 1998; 2005). Nyholm et al., 2004; Walker et al., 2006). In bioluminescence after achieving specific Materials and Methods Anchovy (local name: Mandeli); Indian threshold value (conc. of AI 10 cell mL ¹ in Mackerel (local name: Bangda); and Threadfin brin (local name: Raja Rani) these three fresh marine water fishes were selected culture media) it triggers the synthesis of as a source of bioluminescent bacteria and enzyme luciferase and other enzymes were obtained from fish market near Alpna involved in luminescence (Cao and Meighen Talkies, Camp, Pune. They were kept on ice and soon after brought to the laboratory for further processes. 1989; Rees et al., 1998 Walker et al., 2006; Following fish parts were used as a source for luminescent bacteria (Table 1) (Walker et al., 2006, web links). Berleman, 2009 . This sensing (AI level) Table.1 Fish parts used for luminescent bacteria help to ensure that the luminescence Eye Gill Scales Fin Gut production is enough high to cause an portion region impact in environment and making the Anchovy - Used Used - - process cost effective (Nealson et al., 1970; Mackerel - Used Used - Used Visick et al., 2000). As the production of the Fim Used Used Used Dorsal - luminose enzyme increases ultimately light bream fin generation also increases (Nealson et al., 1970; Cao and Meighen, 1989; Berleman, These different parts were suspended in 2009). sterile sea saline that is 3.5g of sea salt in 100ml distilled water for 24hrs and from this Due to the issues raised by animal rights and loopful suspension was streaked for human rights, the use of microbes like isolation (Web link) on sterile 24 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 23-32 Photobacterium agar (PBA) casein enzyme selective for Vibrios and differential due to hydrolysate 5g/L, yeast extract 2g/L, presence of sucrose and the dyes. TCBS ammonium chloride 0.3g/L, magnesium agar (proteose peptone 10g/L, yeast extract sulfate 0.3g/L, ferric chloride 0.01g/L, 5g/L, sodium thiosuphate 10g/L, sodium calcium chloride 1g/L, potassium phosphate citrate 10g/L, oxagall 8g/L, sucrose 20g/L, (monobasic) 3g/L, sodium glycerophosphate NaCl 10g/L, ferric citrate 1g/L, 23.5g/L, sodium chloride 30g/L, agar 15g/L bromothymol blue 0.40g/L, thymol blue (web link), sea water agar Part A: yeast 0.40g/L, agar 15g/L, pH 8.6±0.2 at 25 C, extract 5g/L, peptic digests of animal tissue formula adjusted, standardized) is highly 5g/L, beef extract 3g/L, agar 15g/L, Part B: selective, meets the nutritional requirements sodium chloride 24g/L, potassium chloride of Vibrio spp., and allows Vibrios to 0.7g/L, magnesium chloride. 6H O 5.3g/L, compete with intestinal flora (Engebrech et magnesium sulfate. 7H O.7g/L, calcium al., 1983; Musa et al., 2008; Ransanga et al., chloride0.1g/L, pH 7.3 +/ 0.2 (web link), 2012; Kannahi and Shivshankari, 2014) Luminus agar (LA) sodium chloride 10g/L, TCBS Agar is recommended by APHA yeast extract 5g/L, peptone10g/L, (American Public Health Association) for agar15g/L, pH 7.3 (web link) and the selective isolation of V. cholerae and V. bioluminescent agar (Boss agar) sodium parahaemolyticus. Enrichment in alkaline chloride30g/L, glycerol1g/L, peptone10g/L, peptone water (M618), followed by isolation meat extract3g/L, pH 7.3 plates (web link). on TCBS Agar is routinely used for isolation Plates were incubated at RT for 48 72hrs of V. cholerae. Vibrios are natural and observed for bioluminescence (web inhabitants of sea water (Kumar, 2010). It is link). possible that a few sucrose-positive Proteus strains can grow to form yellow colonies. Identification of isolated bioluminescent Vibrio parahaemolyticus is a sucrose non- species fermenting organism and produces blue- green colonies, as does Vibrio vulnificus Two isolates were obtained by subsequent (Engebrech et al., 1983; Musa et al., 2008; sub-culturing on sterile LA plates. The Orengo et al., 2010; Ransanga et al., 2012; isolated were named as C1 and C2. After Kannahi and Shivshankari, 2014). morphological colony characterization, Gram staining, motility and Poly- -hydroxy- Triple sugar iron agar (TSI) butyrate staining (by Sudan Black B method) were performed (Bergey s manual TSI is recommended in the identification of 9th edition, web link). Bergey s Manual of gram-negative enteric pathogens in routine determinative Bacteriology 9th edition was examination of faeces. This medium referred and accordingly biochemical tests indicates the ability of an organism to like IMViC, amylase, oxidase, nitrate ferment lactose, sucrose and glucose with reduction were performed and fermentation formation of acid and gas and also its ability of sugars were also checked with 12 to produce hydrogen sulfide gas (Musa et different sugars (Table 2). al., 2008; Kumar, 2010; Ransanga et al., 2012; Kannahi and Shivshankari, 2014) Growth on Thiosulfate Citrate Bile (Table 3 and 4). Sucrose Agar (TCBS) Growth curve TCBS Agar is both differential and highly 25 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 23-32 Loopful of 24 hr old culture of isolate C1 Orange Indicator solution, Conc. and isolate C2 were inoculated in 50 ml Ammonium hydroxide, Potassium Sterile Luminous Broth(LB) and were kept permanganate solution: Dissolve 4 g overnight till absorbance reached 0.1 at 600 KMnO in 100ml water, Diphenylcarbazide nm. After 0.1 absorbance was reached, solution: Dissolve 250 mg in 50ml acetone growth curve experiment was performed by and Store in brown
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